Aureolic acid-type anticancer agents, such as mithramycin (MTM), chromomycin (CMM) and durhamycin (DHM), are potent anticancer and anti-HIV drugs with a unique mode-of-action. They inhibit the growth of cancer cells by cross-linking GC-rich DNA thereby shutting down specificity-protein (Sp)-dependent pathways toward various proto-oncogenes including c-myc and c-src, the latter being associated with the unique hypocalcemic activity found for these drugs. Particularly, MTM is important, and has become a popular biochemical tool to study Sp-dependent signal transduction pathways, but -due to its toxic side effects- is rarely used as anticancer agent, except for the treatment of tumor hypercalcemia refractory to other chemotherapy. However, MTM was identified in 2011 by the NCI as the only lead from a screen of 50,000 compounds that inhibits the EWS-FLI1 transcription factor responsible for the highly malignant phenotype of Ewing sarcomas, which often affect children and were untreatable for the past 40 years. In addition, another NCI group found in June 2012 that MTM represses cigarette smoke induced ABCG2 efflux pumps responsible for the drug resistance of lung and esophageal cancers, and inhibited multiple stem-cell related pathways relevant for tumorigenicity and proliferation of such cancers. A few years ago MTM was identified as a potential lead drug against neurological diseases, arthritis, and for the treatment of hematologic disorders. All these new applications require only very small, less toxic concentrations of the drug, although the mode-of-action in these contexts remains obscure, particularly the mode affecting the EWS-FLI1 transcription factor. MTM's biosynthesis has been studied intensely during the previous funding periods of this research project, and consequently pursued combinatorial biosynthetic efforts revealed various biosynthetic intermediates and many new MTM-analogues, which allowed deducing important structure-activity-relationships. Two regions are of special interest: First, a modification of the 3-side chain, first exemplified in the superior analogues MTM SK and MTM SDK, led to much better anticancer activity profiles with a greatly improved therapeutic index compared to MTM itself. Second, exchange of the D-mycarose sugar in E-position of MTM's trisaccharide chain by D-digitoxose led to superior analogues with greatly reduced toxic side effects. This now allows for new strategies to further concentrate on these regions of the molecule for further drug optimization. During the previous biosynthetic studies biosynthetic intriguing and interesting key enzymes were discovered, which need to be further investigated, particularly the co-dependent enzymes MtmGIV/MtmC (bifunctional glycosyltransferase/methyltransferase-ketoreductase), MtmOIV/MtmW (C-C-bond cleaving oxygenase/ketoreductase), which play key roles for the 3-side chain and the trisaccharide chain formation. Furthermore, the PKS release remains unclear, and it is hypothesized that another co-dependent enzyme pair, namely oxygenase MtmOII and cyclase MtmX play an important role for this process. The goal here is to gain a deeper understanding of the biosynthetic roles, mechanisms and interactions of these key enzymes of the MTM pathway, to pave the way for the optimization and re-engineering of these enzymes that are crucial for novel, further improved MTM derivatives. It is planned to (a) further investigate unclear biosynthetic steps and mechanisms of the MTM and other aureolic acid pathways, (b) to further develop strategies for the selective generation of new, improved MTM analogues including selective 3-side chain and sugar exchange/glycorandomization strategies, (c) to analyze intriguing enzymes, particularly recently discovered co- dependent enzyme pairs. New resulting MTM analogues will be submitted to the NCI DTP (developmental therapeutics program) for further mechanistic investigations.